The prevalence of amputees in the USA is estimated to be 1,1285,000. Recently, hand allo-transplantation became available as a treatment option for amputees. The functional outcome of eight transplanted hands performed worldwide has been remarkably good and is similar to the functional outcome of replanted hands. However, the risk and cost of immuno-suppression have hindered the widespread application of hand allotransplan-tation. In an effort to find an alternative to life-long immuno-suppression, we have successfully demonstrated, in a rat hind limb transplantation model, that donor-specific tolerance can be achieved by inducing mixed allogeneic chimerism (MAC) in the host. To induce tolerance with MAC, three requirements need to be met: 1) donor bone marrow needs to be present as engrafted stem cells in the host, 2) rejection of the donor stem cells needs to be prevented, and 3) graft-vs-host disease (GvHD) needs to be circumvented. The conventional approach to induce MAC is associated with toxic induction protocols, the risk for GvHD, and a delay period between MAC induction and the development of a tolerant state in the host. These obstacles have hindered the clinical applicability of MAC as a valuable alternative for immunosuppressive drugs. The purpose of this proposal is to determine whether we can induce MAC in a clinically relevant manner by taking advantage of the donor bone marrow within a transplanted limb and by using co-stimulatory blockade (a short, term, non toxic immunological therapy that deactivates T cells). In Aim # 1, we will determine whether co-stimulatory blockade therapy can prevent GvHD and acute rejection after limb transplantation. In Aim # 2, we will determine whether the bone marrow within a transplanted limb can induce MAC and tolerance, without conventional immuno-suppression or toxic conditioning of the host. We will use four experimental groups to determine whether co-stimulatory blockade therapy can prevent GvHD and acute rejection of the limb, and another four experimental. groups to determine whether the engrafted stem cells that come within the limb can induce MAC and tolerance to the graft. The significance of this proposal resides in its stated goal of defining the first clinical model for limb transplantation without immuno-suppression. The functional results obtained after limb transplantation with immuno-suppression match those obtained after limb replantation. Therefore, limb transplantation without toxicity to the host will provide a low-risk and inexpensive alternative to the presently used toxic immuno-suppressive drug regimens, offering the best possible prognosis to millions of amputees worldwide.